PROJECT SUMMARY The goal of this proposal is to combine functional mutagenesis, biochemistry, and structural biology, to understand the molecular architecture of the mitochondrial Ca2+ uniporter. Early biochemical studies demonstrated that isolated mitochondria could transport and buffer huge amounts of Ca2+ across their inner membrane via a highly selective, Ru360-sensitive channel called the ?uniporter?. Uptake of Ca2+ via the uniporter is known to activate the TCA cycle, while its overload leads to cell death. Although the uniporter has been studied extensively for over 50 years, its molecular identity remained elusive until our group utilized comparative genomics to discover its molecular components. In humans, the minimal genetic elements required for uniporter current are MCU (the pore forming protein) and EMRE (a single-pass membrane protein). We have recently shown that Dictyostelium discoideum harbors a simpler uniporter, requiring only one component (DdMCU, the homolog of MCU), which is necessary and sufficient for uniporter activity and complements the requirement for MCU or EMRE in human knockout cells. At present, the molecular basis for the uniporter?s selectivity, mechanistic basis for inhibition by Ru360, and why the animal uniporter requires EMRE are unclear. Recently, we have solved the NMR structure of the channel-forming region of MCU using a N-Terminal Domain (NTD) truncated but fully functional construct from C. elegans, named cMCU-?NTD. The structure, which represents a new architecture for Ca2+ channel, provides the much-needed framework for addressing the above questions. Aims 1 and 2 will use DdMCU, which is active by itself, to address the open state of MCU, whereas Aims 3 and 4 will focus on animal MCU and EMRE, to address the mechanism of how EMRE activates the MCU channel. Specifically, in Aim 1, we will perform systematic but hypothesis- driven mutagenesis of DdMCU to identify key residues important for the function and pharmacologic properties of this channel. In Aim 2, we will determine the structure of DdMCU using independent structural approaches including crystallography, EM, and NMR. In Aim 3, we will investigate the biochemistry and topology of EMRE and perform systematic mutagenesis to identify protein regions critical for MCU-EMRE interaction. Finally, in Aim 4, we will investigate Ca2+ and Ru360 binding to MCU using the cMCU-?NTD NMR system, and perform biochemical and structural characterization of MCU-EMRE interaction, including solving the high resolution structure of EMRE alone in lipid bilayer. The proposed studies will yield deep insights into the molecular architecture and mechanisms of the uniporter that promise to have implications for a range of human diseases.